Complete DiGeorge anomaly (cDGA) is a primary immunodeficiency disease caused by a malformation of the third and fourth pharyngeal pouches resulting in congenital athymia and severe T cell deficiency. The treatment for patients with cDGA has been largely limited to transplantation of intact slices of postnatal thymus removed from immunologically normal infants. This approach has produced clinical responses but results remain suboptimal; nave T cell numbers persist indefinitely below the 10th percentile and autoimmune disease is common. Thus there is an urgent need to improve the outcome for this lethal condition. The overall goal of this proposal is to generate thymic organoids that can provide long term production of functional and tolerant T cells after transplantation into patients with cDGA. We hypothesize that the suboptimal immunologic and clinical outcomes with the current transplantation approach are at least partly due to poor survival and function of the thymic microenvironment i.e. thymic epithelial cells (TECs) and thymic mesenchymal cells (TMCs), during ex vivo culture. In addition, we propose that delayed vascularization of the thymic slices after implantation results in further damage to TECs and delayed seeding of hematopoietic stem and progenitor cells (HSPC) from the bone marrow. We have developed methods to generate implantable, three-dimensional human thymic organoids consisting of ex-vivo expanded, human postnatal TECs and TMCs, depleted of endogenous thymocytes. This approach allows gene expression and cell composition to be manipulated prior to re-aggregation with human HSPC into organoids. After implantation in immunodeficient mice, functional T cells with a diverse T cell repertoire are generated from human HSPCs within the organoids, and also from HSPC that migrate from the bone marrow. Expression of Vascular Endothelial Growth Factor (VEGF) in TMCs significantly improves vascularization, TEC survival and thymocyte production after implantation. In an athymic, congenic mouse model, we find that thymic organoids induce robust production of conventional and regulatory T cells. This proposal is designed to address specific issues that we believe are critical to understand and develop the clinical potential of thymic organoids to improve outcomes for patients with cDGA: 1: To optimize survival and function of the thymic microenvironment after implantation We will define parameters for two key factors that we hypothesize determine how well thymic organoids survive and function after transplant: duration of TEC culture before implantation and VEGF expression after implantation 2. To determine the ability of allogeneic donor thymic organoids to generate tolerant host T cells Using an allogeneic nude mouse model, we will test the ability of thymic organoids to produce T cells from host HSPC that are tolerant to self and to donor.